© 2017. Published by The Company of Biologists Ltd | Journal of Experimental Biology (2017) 220, 3556-3564 doi:10.1242/jeb.155440

RESEARCH ARTICLE Physiological mechanisms constraining ectotherm fright-dive performance at elevated temperatures Essie M. Rodgers and Craig E. Franklin*

ABSTRACT such as locomotor performance (Johansen and Jones, 2011), Survival of air-breathing, diving ectotherms is dependent on their developmental rates/growth (McLeod et al., 2013), immune capacity to optimise the time available for obligate underwater competence (Yu et al., 2009) and survival (Rohr and Palmer, 2013). activities, such as predator avoidance. Submergence times are The consequences of elevated temperatures on ectotherm thermally sensitive, with dive durations significantly reduced by performance are well documented (Bellard et al., 2012; increases in water temperature, deeming these animals particularly Kingsolver et al., 2013) but the physiological basis for loss of vulnerable to the effects of climate change. The physiological performance remains unclear. Performance decrements at mechanisms underlying this compromised performance are unclear stressfully high temperatures are hypothesised to stem from but are hypothesised to be linked to increased oxygen demand and a oxygen demand exceeding oxygen supply capacity [i.e. cardio- reduced capacity for metabolic depression at elevated temperatures. respiratory system failure; oxygen- and capacity-limited thermal Here, we investigated how water temperature (both acute and chronic tolerance (OCLTT) hypothesis] (Pörtner, 2001, 2010; Pörtner and exposures) affected the physiology of juvenile estuarine crocodiles Knust, 2007; Pörtner and Farrell, 2008; Eliason et al., 2011). Compromised aerobic performance may occur at high temperatures (Crocodylus porosus) performing predator avoidance dives (i.e. ̇ ‘ ’ when maximal rates of oxygen consumption (VO ,max) plateau or fright-dives). Diving oxygen consumption, fright bradycardia, ̇ 2 haematocrit and haemoglobin (indicators of blood oxygen carrying decrease but resting/standard metabolic rate (VO2,standard) increases ̇ − ̇ capacity) were assessed at two test temperatures, reflective of exponentially, reducing absolute aerobic scope (=VO2,max VO2,standard). different climate change scenarios (i.e. current summer water A narrowed aerobic scope is thought to translate into a reduced temperatures, 28°C, and ‘high’ climate warming, 34°C). Diving capacity for activities including growth, movement, digestion and oxygen consumption rate increased threefold between 28 and 34°C reproduction (Pörtner, 2002), and the thermal effects on individuals may scale up to affect population and community dynamics (Pörtner (Q10=7.4). The capacity to depress oxygen demand was reduced at elevated temperatures, with animals lowering oxygen demand from and Peck, 2010). A narrowed aerobic scope at high temperatures has surface levels by 52.9±27.8% and 27.8±16.5% (means±s.e.m.) been demonstrated in a number of tropical ectotherms (Munday at 28°C and 34°C, respectively. Resting and post-fright-dive et al., 2009; Nilsson et al., 2009; Johansen and Jones, 2011; haematocrit and haemoglobin were thermally insensitive. Together Rummer et al., 2014); however, the OCLTT hypothesis is not these findings suggest decrements in fright-dive performance at universal (Clark et al., 2013; Ern et al., 2014, 2015, 2016; Norin elevated temperatures stem from increased oxygen demand coupled et al., 2014). Many ectotherms experience compromised with a reduced capacity for metabolic depression. performance at temperatures below those affecting aerobic scope (Norin et al., 2014), suggesting the mechanistic explanation may be KEY WORDS: Aerobic dive limit, Diving metabolism, Thermal multi-faceted and thus it remains unresolved in many taxa. sensitivity, Climate change, Bradycardia, Heart rate Air-breathing, diving ectotherms (e.g. sea snakes, marine iguanas, turtles and crocodylians) appear vulnerable to increases INTRODUCTION in water temperature as dive capacity (i.e. time spent submerged/ The performance and survival of many ectothermic species is dive duration) is inversely related to water temperature (Fuster et al., predicted to be compromised under global climate change (Pörtner 1997; Prassack et al., 2001; Priest and Franklin, 2002). Shortened and Farrell, 2008). Altered thermal regimes are particularly dive times translate into less time available for obligate underwater threatening to ectotherms (almost all fish, amphibians and activities such as foraging/hunting, predator avoidance, sleep/rest reptiles) as body temperature is closely tied to the thermal and social interactions. Submergence times of free-ranging environment. Ectotherm performance is optimised within a crocodylians and turtles are reduced in summer months compared limited range of body temperatures (i.e. thermal performance with winter months (Carr et al., 1980; Bentivegna et al., 2003; breadth) and ongoing climate warming will probably drive Gordos et al., 2003; Hochscheid et al., 2005; Bradshaw et al., 2007; temperatures beyond sustainable limits (Rummer et al., 2014). Campbell et al., 2010a), suggesting these animals are not fully Body temperatures surpassing thermal performance optima are compensating for present-day seasonal thermal fluctuations. Dive generally accompanied by a marked decline in fitness-related traits durations are predicted to be further reduced by ongoing increases in water temperature in marine and freshwater habitats, with submergence times of some species forecasted to halve under a School of Biological Sciences, The University of Queensland, St Lucia, Brisbane, QLD 4072, Australia. moderate rate of climate warming (SRES A1B storyline; 50th percentile of IPCC global warming range; Rodgers et al., 2015). A *Author for correspondence ([email protected]) limited or non-existent capacity to thermally acclimate/acclimatise C.E.F., 0000-0003-1315-3797 to elevated temperatures following long-term (i.e. chronic) exposure elevates the susceptibility of this group to climate

Received 22 December 2016; Accepted 25 July 2017 change (Clark et al., 2008; Rodgers et al., 2015), but physiological Journal of Experimental Biology

3556 RESEARCH ARTICLE Journal of Experimental Biology (2017) 220, 3556-3564 doi:10.1242/jeb.155440

animal escapes/dives underwater in response to a perceived threat List of abbreviations (e.g. loud noise or the presence of an experimenter; Wright et al., ADL aerobic dive limit 1992). Diving is a vital pre-condition for the onset of fright fH heart rate bradycardia and heart rate does not drop in C. porosus in response to OCLTT oxygen- and capacity-limited thermal tolerance a threat on land (Wright et al., 1992). The mechanism underlying the RQ respiratory quotient thermal sensitivity of fright-dive performance probably differs TBO total body oxygen ̇ from OCLTT, which does not consider hypometabolic states. High VO oxygen consumption rate ̇ 2 VO ,dive diving oxygen consumption rate temperatures may not only elevate fright-dive metabolic rate ̇ 2 ̇ ’ VO2,post-dive post-fright-dive oxygen consumption rate (VO ,dive) but also compromise a diver s capacity to depress ̇ 2 VO2,pre-dive surface/pre-fright-dive oxygen consumption rate metabolic rate from resting/surface levels. V̇ standard oxygen consumption rate O2,standard The aim of this study was to investigate how the fright-dive physiology of juvenile estuarine crocodiles is affected by increases in water temperature. Crocodylians are primarily aquatic, and free- mechanisms constraining performance at high temperatures ranging animals have been recorded to dive 50–70 times per day remain uncertain. (Campbell et al., 2010a). The ability to remain submerged for The aerobic dive limit (ADL) conceptually represents the extended periods of time is thought to be adaptive because predator maximum duration an animal can remain submerged before avoidance, foraging, sleep/recovery and social interactions occur oxygen debt is incurred (Butler, 2006). An individual’sADLis underwater (Seebacher et al., 2005; Campbell et al., 2010b). dependent on total body oxygen (TBO) stores and the rate at which Predator escape dives are vital in hatchling and juvenile C. porosus, these stores are consumed, with smaller stores and/or a faster rate of which may fall prey to a range of species including: monitor lizards oxygen consumption reflective of a shorter ADL (Butler, 2006). (Varanus mertensi, Varanus panoptes), barramundi (Lates Oxygen can be stored in the lungs, blood and tissue, and the calcarifer), whistling kites (Haliastur sphenurus), olive pythons percentage contribution of stores varies between species (Kooyman, (Liasis olivasceus), great egrets (Ardea alba) and larger C. porosus 1989). In estuarine crocodiles (Crocodylus porosus), for instance, (Grigg and Kirshner, 2015). The fright-dive capacity of juvenile pulmonary (i.e. lung) oxygen represents the majority of stores C. porosus is thermally sensitive, with marked reductions in dive (∼67.0%), followed by blood oxygen (∼28.9%), and tissue oxygen duration at temperatures above 28°C (Rodgers et al., 2015), but the contributes the least (∼4.1%) (Wright, 1985). Ectotherm oxygen physiological mechanism underlying this pattern is unresolved. We stores decline with increasing temperature (Pough, 1976; Fuster et al., assessed the effect of water temperature on V̇ and fright ̇ O2,dive 1997); however, dive duration is reduced at elevated temperatures bradycardia. We hypothesised that VO2,dive would increase beyond the extent expected from reduced TBO stores alone (Hayward exponentially with temperature and align with compromised et al., 2016). The thermal sensitivity of ectotherm diving performance performance (i.e. shorter dive duration; hypothesis 1). Similarly, is hypothesised to be linked to a reduction in the ADL as a result of we predicted that C. porosus’ relative capacity for metabolic diving metabolic rate (i.e. oxygen consumption) increasing depression (i.e. reductions in heart rate and oxygen consumption exponentially with rising water temperature (Hayward et al., 2016). compared with surface rates) would be impaired at elevated The validity of this hypothesis remains untested in crocodylians, but temperatures (hypothesis 2). The assessment of fright-dive shorter dive durations at elevated temperatures have been associated physiology was subsequently used to uncover the mechanisms with increased diving metabolism in two species of sea snake (spine- underlying impaired performance at elevated temperatures. bellied sea snake, Hydrophis curtus, and elegant sea snake, Hydrophis elegans; Udyawer et al., 2016). Ectotherm divers MATERIALS AND METHODS routinely dive within ADLs but submergences can be extended Animal maintenance beyond this limit with the use of anaerobic pathways (Butler and Estuarine crocodiles (Crocodylus porosus; Schneider 1801) were Jones, 1982; Seymour, 1982). Exceeding the ADL incurs the cost of obtained from two sources; eggs were collected from a single clutch longer post-dive surface intervals to clear accumulated lactate at David Fleay Wildlife Park (Burleigh Heads, QLD, Australia, (Kooyman et al., 1980; Costa et al., 2004). −28.108901, 153.444175) and juveniles were obtained from three Vertebrates enter a unique physiological state when diving, with a clutches at Cairns Crocodile Farm (Gordonvale, QLD, Australia, suite of cardiovascular alterations occurring (Andersen, 1966). This −17.039566, 145.792283; N=14 from 4 clutches: 6 reared from state is termed the ‘dive response’ and includes a decrease in heart eggs, 8 obtained as juveniles). Eggs were transported to The rate (i.e. diving bradycardia), a redistribution of blood stores to University of Queensland (St Lucia, QLD, Australia), where they essential organs (i.e. peripheral vasoconstriction) and a cardiac were incubated in an R-com 50 egg incubator (Auto Elex Co. Ltd, shunt (in some species; Blix and Folkow, 1983; Butler and Jones, GimHae, Korea) for 85 days at 31.5±1°C and 70–90% humidity. 1997). These alterations may facilitate prolonged submergence by Upon hatching, animals were maintained in an environment aimed lowering oxygen demands, and animals enter a hypometabolic state at optimising healthy growth for 12 months prior to testing (water where oxygen demands are lower than surface metabolic rates temperature 31.5±0.5°C). The juveniles obtained from Cairns (Davis et al., 2004; Hastie et al., 2007). The initiation of the dive Crocodile Farm had been incubated at 32.5±0.5°C and kept in response appears to be context specific in some species (Gaunt and outdoor enclosures (i.e. March 2014 to June 2015) before we Gans, 1969; Noren et al., 2012); with crocodylians, for example, received them. Crocodiles were fed regularly (twice weekly, markedly reducing heart rate (65±6% reduction) during predator totalling 15% of their body mass) a mixture of minced beef, avoidance dives (i.e. fright-dives) and only small cardiovascular chicken and pilchards supplemented with powdered calcium and changes (14±6% reduction) are observed during voluntary, vitamin D (Vetafarm, Wagga Wagga, NSW, Australia). Enclosures undisturbed dives (Wright et al., 1992). A pronounced drop in were cleaned, with complete water changes after feeding. All heart rate during a predator avoidance dive is termed ‘fright animals were acclimated to a common water temperature of 31.5°C bradycardia’ and can be initiated in a laboratory setting where an for 3 months prior to testing, to counteract differences in thermal Journal of Experimental Biology

3557 RESEARCH ARTICLE Journal of Experimental Biology (2017) 220, 3556-3564 doi:10.1242/jeb.155440 history. Animals were between 15 and 22 months old at the time of Fright-dive performance was assessed in animals from both testing (total length, 56.6±9.6 cm; snout–vent length, 28.9±5.2 cm; thermal acclimation treatments (i.e. 28°C acclimated and 34°C body mass, 488.0±251.6 g; means±s.d.). All experiments complied acclimated) at two test temperatures (28 and 34°C). Position in the with The University of Queensland animal ethics requirements dive tank (i.e. partition assignment) and the order of test temperature (approval no. SBS/018/14/ARC/AUST ZOO). were randomised. Animals were given an extended habituation period (minimum of 8 h) to ensure full recovery after handling stress Experimental design and thermal acclimation treatments (Franklin et al., 2003). Following the habituation period, either a Crocodiles were assigned to one of two thermal acclimation single fright-dive trial or a sustained fright-dive trial began. The treatments, based on the following Intergovernmental Panel on single fright-dive condition involved just one dive, whereas the Climate Change (Solomon et al., 2007) climate change scenarios. sustained fright-dive condition involved a bout of four consecutive (1) Low rate of global warming (SRES B1 storyline; 10th percentile dives (i.e. sustained diving) with fixed surface intervals of 3.6± of IPPC global warming range)/current summer water temperature. 0.6 s. Fright-dives were designed to simulate the presence of a This scenario reflects a low-emissions future. Summer water predator where crocodiles dived underwater as an escape behaviour. temperatures remain unchanged in this scenario. Experimental Fright-dives were initiated by an experimenter lightly tapping a water temperature simulating this scenario was 28±0.5°C (N=7). crocodile with a blunt wooden pole. The experimenter was in view (2) High rate of global warming (SRES A1F1 storyline; 90th of the crocodile throughout the dive trial to simulate the prolonged percentile of IPCC global warming range). This scenario is based on presence of a predator. Dive durations were directly observed and the intensive and continued use of fossil fuels, with unprecedented timed. levels of carbon emissions, population growth and industrial expansion. Summer water temperatures are predicted to range Blood sampling and analyses between 33 and 35°C. Experimental water temperature simulating Crocodiles were immediately captured upon surfacing at the end of this scenario was 34±0.5°C (N=7). a fright-dive trial for blood sampling. Blood samples (0.5–2ml) Treatment assignment was randomised using a random were drawn from a branch of the jugular vein (venous blood) using number generator (www.random.org; even number→28°C, odd ½ in 23 gauge needles attached to heparinised (lithium salt; Sigma- number→34°C) but occurred separately for crocodiles from each Aldrich Sydney, NSW, Australia) syringes. An aliquot (∼5µl)of source (i.e. Cairns Crocodile Farm and David Fleay Wildlife Park) whole blood was used to determine haemoglobin concentration to ensure animals from the two sources were equally distributed ([Hb]) and two microcapillary tubes were filled to measure between treatments. Thermal acclimation treatments were identical haematocrit (percentage packed red cell volume, %Hct). apart from water temperature and enclosures were large wooden Microcapillary tubes were spun at 5000 g for 2 min (micro- tanks (3.35 m×0.85 m×0.75 m, length×width×height) designed to haematocrit centrifuge, Hawksley, Lancing, Sussex, UK) and %Hct emulate thermally heterogeneous environments conducive to was measured. A colorimetric assay kit (Sigma-Aldrich; MAK115) thermoregulatory behaviour. Water temperatures were maintained was used to determine duplicate [Hb]. Blood samples were also using 300 W submersible heaters attached to thermostats taken from resting animals as a reference point. Resting blood (Aquasonic, Wauchope, NSW, Australia). Enclosures contained samples were obtained by capturing animals in their holding tank freshwater filled to a depth of 0.15 m. Dry platforms were situated at and immediately sampling. Blood sampling times were recorded each end of the tanks, one being a relatively ‘warm’ platform and all samples were obtained within 3 min (Finger et al., 2015a,b). situated underneath a ceramic heat lamp (250 W; OzWhite, Enfield, SA, Australia; suspended 26 cm above the platform) and a UV-B Fright bradycardia ® −1 light (25 W; Exo Terra , Montreal, QC, Canada) and the other a Surface and fright-dive heart rates ( fH; beats min ) were measured relatively ‘cool’ platform with no lamps. Basking opportunity (i.e. in crocodiles from both acclimation treatments (28°C acclimated the time the heat lamp was switched on) was 8 h day−1 (08.00 h– and 34°C acclimated) at two test temperatures (28 and 34°C). 16.00 h) for all treatments, with substrate temperature underneath Animals were placed in a plastic dive tank (37×39×56 cm, the heat lamp averaging 40±3°C (mean±s.d.). A summer length×width×height) containing freshwater (depth 26 cm) within photoperiod was used, with a constant 14 h:10 h light:dark a temperature-controlled room. Two electrocardiogram (ECG) wires regimen (05.00 h–19.00 h light) for all treatments. Animals were (MLA1203 Needle electrode, AD Instruments, Sydney, NSW, left to acclimate to thermal treatments for 60 days prior to Australia) were inserted subcutaneously on the animal’s ventral performance testing and were fasted for 48 h prior to dive trials. surface, anterior and posterior to the heart, and held in place using strapping tape (Elastoplast rigid sports strapping, Beiersdorf, Fright-dive trials Hamburg, Germany). The ECG wires were run into an adjacent Dive trials were held in a large experimental tank (1.8 m× room, attached to a BioAmp (ML132, ADInstruments Pty Ltd, 2.0 m×1.9 m, length×width×height) custom built from foam Bella Vista, NSW, Australia) and the bioamp was connected to a fibreglass. The dive tank was evenly partitioned into three sections PowerLab (4/30 series ML866, ADInstruments). fH recordings were with opaque plastic partitions allowing three dive trials to run visualised on a laptop using LabChart software (ADInstruments) concurrently. The dive tank contained filtered freshwater to a depth of with a sampling rate of 100 Hz. Avideo camera (Microsoft LifeCam 1.3 m and water temperature was finely controlled using a spa heater Studio, Microsoft, Redmond, WA, USA) was placed above the dive (900 EVO, Elecro Engineering, Stevenage, UK). Thermal profiling tank and recordings were synchronised with fH readings so that dive of the dive tank using Thermocron temperature loggers (iButtonLink events could be easily isolated. Three fright-dives were initiated by Technology, Whitewater, WI, USA) confirmed the uniformity of the experimenter entering the room and lightly touching the animal. temperature throughout the water column. Each partitioned section of Animals were allowed to rest for 30 min between each fright-dive. the dive tank contained a floating rest platform (0.6 m×0.15 m× Surface and fright-dive fH were determined by extracting and 0.05 m, length×width×height), where crocodiles could rest and averaging multiple recordings (three per animal) between 60 and breathe on the water surface whilst their body remained submerged. 300 s in duration. Surface fH was recorded before each fright-dive. Journal of Experimental Biology

3558 RESEARCH ARTICLE Journal of Experimental Biology (2017) 220, 3556-3564 doi:10.1242/jeb.155440

Fright-dive and surface fH were averaged for each animal at each test Statistical analyses temperature. Relative (%) fright bradycardia was calculated as: Data analyses were performed in R Studio (version 3.1.3; www.R- project.org/) using the nlme (linear and non-linear mixed effects % ¼ð Þ= ; ð Þ Bradycardia fH;surface fH;diving fH;surface 100 1 models; https://CRAN.R-project.org/package=nlme) package. A series of linear mixed effects models were used to determine the where fH,surface is mean heart rate when the animal is at the surface of effects of test temperature and thermal acclimation treatments on the water and fH,diving is mean heart rate when the animal is fright-dive performance (i.e. minutes submerged), fright-dive performing a fright-dive. Surface fH measurements were not a ̇ metabolism (VO ,dive) and fright-dive fH (% fright bradycardia). measure of resting fH but a measure of routine surface fH. Animals 2 were left to recover after instrumentation and equilibrate body Test temperature (two-level factor), acclimation treatment (two- temperature with water temperature for a minimum of 1 h before level factor), body mass and dive tank partition number were included as fixed effects and animal source (i.e. David Fleay trials began, at which point fH recordings had levelled out. Wildlife Park or Cairns Crocodile Farm) and identification number Fright-dive metabolism (ID) were included as random effects (ID nested within source) in all ̇ models. Statistical significance was accepted at P≤0.05. Fright-dive metabolic rate (VO2,dive) was determined for crocodiles from each thermal acclimation treatment at two test temperatures (28 and 34°C) using flow-through respirometry. Animals were fasted RESULTS for a minimum of 5 days prior to testing to eliminate metabolic Fright-dive performance responses to feeding (i.e. specific dynamic action; Gienger et al., Single and sustained fright-dive performance (i.e. total time spent 2011), and left to adjust to dive tank conditions for 1 h prior to submerged during four consecutive dives) were thermally sensitive, testing to ensure body temperature had equilibrated with water with performance decrements experienced at test temperatures ̇ of 34°C compared with 28°C (single P<0.05, F1,9=19.2, temperature. VO2,dive was measured inside a custom-built diving column (height 1.33 m, base diameter 0.42 m, top diameter 0.25 m) lme; sustained P<0.0001, F1,10=59.9, lme; Fig. 1A,B). Single placed inside the dive tank at a water depth of 1.3 m. The water submergences performed at 28°C lasted 18.5±2.4 min (pooled surface was sealed using a custom-fitted piece of Styrofoam with a mean±s.e.m.) and were reduced to 9.0±1.0 min (pooled mean± dome-shaped respiratory hood (volume 3.6 l) fitted with inflow and s.e.m.) at 34°C (Q10=0.30; Fig. 1A). Similarly, crocodiles diving at outflow air outlets. The diving column and respiratory hood were 28°C spent an average of 66.7±5.9 min (pooled mean±s.e.m.) designed to ensure the only available air space was inside the underwater during sustained dive trials, whereas animals diving at respiratory hood. A pull-system was utilised with flow rate ranging 34°C spent an average of 28.2±1.9 min (pooled mean±s.e.m.) between 590 and 1240 ml min−1, depending on animal body submerged (Q10=0.24; Fig. 1B). Both sustained and single mass, using a mass-flow controller (SS3, Sable Systems diving performances were independent of thermal acclimation International, North Las Vegas, NV, USA; calibrated with a treatment, with no observed differences in C. porosus acclimated Bubble-O-Meter, Dublin, OH, USA). Outflowing air was scrubbed to 28°C compared with 34°C (single P=0.45, F1,11=0.6, lme; of water vapour by passing it through a drying column (Drierite, sustained P=0.87, F1,10=0.03; Fig. 1A,B). Covariate interactions among body mass (P≥0.91) and partition number (P≥0.70) were Sigma-Aldrich). Fractional concentrations of CO2 and O2 were not significant. measured by passing dry air into a CO2 analyser (LI-820, LI-COR, Lincoln, NE, USA) and subsequently into an O2 analyser (Oxzilla, Fright bradycardia Sable Systems International). The CO2 meter malfunctioned beyond correction for many trials, so rates of O consumption fH,surface was significantly higher at a test temperature of 34°C 2 −1 −1 ̇ −1 (65±4 beats min ) than at 28°C (46±3 beats min ; Fig. 1C; (VO2, ml min ) were calculated using eqn 11.2 from Lighton (2008) and respiratory quotients (i.e. ratio of carbon dioxide produced to P<0.001, F1,6=43.7, lme) but independent of thermal acclimation oxygen consumed at a given time point) were estimated for each treatment (P=0.54, F1,5=0.4, lme). Fright-dive fH ( fH,diving)was test temperature using Grigg’s (1978) eqn 2 derived from juvenile independent of thermal acclimation treatment (P=0.30, F5,1=1.3, C. porosus (N=11; P<0.001): lme) but test temperature had a borderline significant effect (P=0.052, F1,6=5.8, lme), with elevated fH,diving at 34°C RQ ¼ 1:098 0:0203T; ð2Þ (24±2 beats min−1) compared with 28°C (19±1 beats min−1; Fig. 1C). All animals exhibited fright bradycardia, with fH reduced where RQ is the respiratory quotient and T is test temperature (°C). −1 ̇ by 12–55 beats min from surface levels. Relative fright bradycardia Surface oxygen consumption rates (VO2,pre-dive) were measured for (i.e. percentage reduction from fH,surface) was thermally insensitive 1 h, followed by a single fright-dive. Post-fright-dive oxygen (test temperature P=0.44, F =0.3; acclimation treatment P=0.65, consumption (V̇ ) was measured to estimate oxygen debt 1,6 O2,post-dive F =0.2, lme; Fig. 2A). Body mass had no significant effect on accumulated during submergence, and oxygen debt was assumed 1,5 ̇ ̇ ̇ fH,surface, fH,diving and relative fright bradycardia (P>0.27, lme). to be cleared once VO2,post-dive equalled VO2,pre-dive. VO2,dive was calculated using Eqn 3 (Hurley and Costa, 2001): Fright-dive metabolism _ _ Surface (V̇ ) and fright-dive (V̇ ) metabolic rates were V ; ¼ V ; =DD; ð3Þ O2,pre-dive O2,dive O2 dive O2 debt significantly higher at a test temperature of 34°C than at 28°C ̇ ̇ ̇ where VO2,debt represents oxygen debt accumulated during (Fig. 1D; VO2,pre-dive P<0.01, F1,5=19.1, Q10=2.32, lme; VO2,dive submergence (ml min−1) and DD is dive duration (i.e. total time P<0.05, F =9.1, Q =7.4, lme). Thermal acclimation treatment 1,4 10 ̇ submerged, min). Animals were motionless during surface periods and body mass had no effect on VO2,pre-dive (thermal acclimation and dives (excluding ascending and descending movements). treatment P=0.73, F1,5=0.1; body mass P=0.16, F1,5=0.2, lme) and Baseline measurements (in the absence of animals) were taken V̇ (thermal acclimation treatment P=0.37, F =0.98, body O2,dive ̇ 1,5 before and after trials for a minimum of 2 h to detect drifts in mass P=0.13, F1,4=3.5 lme). VO2,dive was depressed from surface ambient fractional concentrations of O2 and CO2. levels by 52.9±27.8% at 28°C and 27.8±16.5% at 34°C (pooled Journal of Experimental Biology

3559 RESEARCH ARTICLE Journal of Experimental Biology (2017) 220, 3556-3564 doi:10.1242/jeb.155440

A B 40 28°C acclimated 120 28°C acclimated 35 34°C acclimated a a 100 34°C acclimated a a 30 80 25 20 60 b b b 15 b 40 10

Dive duration (min) 20 5

0 time submerged (min) Total 0 28 28 34 34 28 28 34 34

D 80 C Surface 3.0 Surface Diving Diving d ) 2.5 –1 ) 60 c –1 kg 2.0 c

c –1 40 1.5 b

(ml min 1.0 (beats min b 2 H O f 20 a . a V 0.5

0 0 28 34 28 34 Test temperature (°C) f V̇ Crocodylus Fig. 1. Thermal sensitivity of fright-dive performance, heart rate ( H) and oxygen consumption rate ( O2) in juvenile estuarine crocodiles ( porosus). Both single (A) and sustained (B) fright-dive performance, assessed as minutes submerged, was reduced at a test temperature of 34°C compared with 28°C (single P<0.05; sustained P<0.0001; linear mixed-effects model, lme; N=6–7 per acclimation treatment; Table S1), regardless of thermal ̇ acclimation treatment (P≥0.52, lme). Surface and diving fH (N=4 per acclimation treatment; C) and VO (N=3–4 per acclimation treatment; D; Table S1) increased ̇ 2 ̇ with rising temperature ( fH,surface P<0.001, lme; fH,diving P=0.052, lme; VO2,pre-dive P<0.01, Q10=2.32, lme; VO2,diving P<0.05, Q10=7.4, lme). Values are shown as raw data points in A and B, and as means±s.e.m. in C and D. Different letters indicate significant differences between groups. means±s.e.m.). Test temperature had a borderline significant effect Hct and Hb −1 on relative metabolic depression (Fig. 2B; P=0.052, F1,4=6.8, lme) Resting Hct (%) and [Hb] (g l ) were independent of thermal but was independent of thermal acclimation treatment (P=0.32, acclimation treatment (Hct P=0.49, F1,8=0.5; [Hb] P=0.58, F1,5=0.7, lme) and body mass (P=0.40, F1,4=4.2, lme). Post-fright- F1,5=0.4, lme), with Hct averaging 18±1% and [Hb] averaging dive oxygen debt and recovery duration were thermally insensitive, 64±6 g l−1 in all animals. Post-fright-dive Hct and [Hb] were also averaging 13.5±2.6 ml kg−1 and 18.0±2.0 min for both test independent of both thermal acclimation treatment (Hct P=0.43, temperatures (Fig. 3; O2 debt P=0.07, F1,5=5.7, lme; recovery F1,5=0.7; [Hb] P=0.85, F1,5=0.04, lme) and test temperature (Hct duration P=0.21, F1,4=2.2, lme), and were independent of thermal P=0.40, F1,4=0.9; [Hb]; P=0.74, F1,3=0.1, lme). There was no acclimation treatment (O2 debt P=0.14, F1,5=3,1, lme; recovery change in Hct or [Hb] between resting and post-dive states (Hct duration P=0.29, F1,5=1.4, lme) and body mass (O2 debt P=0.13, P=0.53; [Hb] P=0.97, lme). Blood sampling time had no effect on F1,4=3.8, lme; recovery duration P=0.1, F1,4=2.4, lme). Hct or [Hb] (Hct P=0.11, F1,4=4.1; [Hb] P=0.19, F1,3=2.8, lme).

80 A 80 B a a 70 70 a 60 60

50 50 b 40 40 30 30 depression (%) 2

20 O 20 . V 10 10 Relative bradycardia (%) 0 0 28 34 28 34 Test temperature (°C)

Fig. 2. Fright-dive bradycardia and metabolic depression in C. porosus. (A) Thermal sensitivity of relative ‘fright’ bradycardia (i.e. percentage reduction from fH, surface) of juvenile estuarine crocodiles (N=8 per test temperature, 4 per acclimation treatment). Relative bradycardia was independent of test temperature ̇ – (P=0.44, lme). (B) Effect of test temperature on diving metabolic depression (i.e. percentage reduction from VO2,pre-dive) in juvenile estuarine crocodiles (N=7 8 per test temperature, 3–4 per acclimation treatment; Table S1). Values are shown as individual data points in A and as means±s.e.m. in B. Diving metabolic depression was reduced at a test temperature of 34°C compared with that at 28°C (P=0.052, lme). Different letters indicate significant differences between groups. Journal of Experimental Biology

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10 A submergence. Rapid use of TBO stores probably decreases dive a duration and increases the frequency at which aerially respiring 8 divers must surface and replenish oxygen stores. Diving metabolic rates were more thermally sensitive than surface

) ̇ ̇ 6 metabolic rates (i.e. VO2,dive Q10=7.4; VO2,pre-dive Q10=2.32) and this is –1 a probably due to a compromised capacity for metabolic depression at 4 the 34°C. Diving oxygen requirements were lowered from surface (ml kg levels by 52% and 28% at 28 and 34°C, respectively. A reduction 2 in relative metabolic depression suggests the capacity for hypometabolism is compromised beyond the extent to be expected Post-fright-dive oxygen debt 0 from Q10 effects only. The molecular and biochemical mechanisms 28 34 responsible for initiating the dive response (i.e. metabolic depression) 25 B are not well understood, particularly in ectotherms (Withers and a Cooper, 2010). Diving bradycardia in reptiles is controlled by 20 a interactions between the cholinergic and adrenergic nervous systems governing pacemaker heart cells (Burggren, 1987; Hicks and Farrell, 15 2000; Hicks, 1994). Stimulation of cold receptors (particularly those located in facial regions), coupled with inputs from nasal, lung and (min) 10 carotid chemoreceptors appear to be key in the initiation of the dive response (Drummond and Jones, 1979; Alboni et al., 2011). Cold 5 receptors are unlikely to receive stimulation at elevated temperatures and may underlie the elevated f observed in C. porosus at 34°C. 0 H Post-fright-dive recovery duration 28 34 Further to this, chemoreceptor functioning (e.g. intrapulmonary and Test temperature (°C) brainstem) can be dependent on body temperature in lizards (Douse and Mitchell, 1988; Zena et al., 2016), and it is possible that Fig. 3. Thermal sensitivity of post-fright-dive oxygen debt and recovery chemoreceptor functioning was compromised in C. porosus diving at C. porosus duration in juvenile estuarine crocodiles ( ). N=8 per test 34°C. Crocodiles were able to maintain significant declines in f temperature, pooled from thermal acclimation treatments. (A) Post-fright-dive H oxygen debt (N=3–4, Table S1). (B) Recovery duration (i.e. time required for during submergence (i.e. percentage bradycardia) at both test ̇ ̇ – temperatures, suggesting other components of the dive response VO2,post-dive= VO2,pre-dive, N=3 4; Table S1). Test temperature had no effect on post-fright-dive oxygen debt (P=0.07, lme) and recovery duration (P=0.21, were compromised. For example, limited capacity for the initiation of lme). Different letters indicate significant differences between groups. the dive response, peripheral vasoconstriction (Altimiras et al., 1998) or cardiac shunting may have contributed to reduced metabolic DISCUSSION depression at 34°C, but further experimentation is required for Ectotherm functioning and performance are often compromised at confirmation. elevated temperatures akin to forecasted climate change (Pörtner and Farrell, 2008), but the underlying mechanisms are frequently Hct and Hb unresolved. Here, we partially illuminated the physiological Chronic increases in temperature have elicited compensatory responses mechanisms constraining fright-dive capacity at elevated in ectotherms, whereby the carrying capacity of blood is augmented by temperatures in juvenile estuarine crocodiles. In line with previous increased [Hb] and Hct (Houston and Cyr, 1974; Gallaugher and findings (Rodgers et al., 2015), fright-dive performance of Farrell, 1992; Jayasundara and Somero, 2013; Lilly et al., 2015). In C. porosus was markedly compromised at elevated temperatures, contrast to these findings, [Hb] and Hct remained unchanged in with submergence times halving between 28 and 34°C. juvenile C. porosus following long-term exposure to an elevated Performance decrements appear to be linked to reductions in temperature (i.e. 34°C), suggesting a lack of plasticity. An inability to ADLs at elevated temperatures, stemming from increased oxygen adjust [Hb] and Hct may underlie the absence of thermal acclimation demand and a reduced capacity for metabolic depression in fright-dive performance. However, blood oxygen stores only (supporting hypothesis 1 and hypothesis 2). Cumulatively, these account for approximately one-third (28.9%; Wright, 1985) of TBO results suggest C. porosus terminated dives earlier at the elevated stores, while pulmonary oxygen accounts for ∼67%; therefore, a lack ̇ temperature because of a faster use of body oxygen stores. of compensation in VO2,dive probably played a greater role in compromised fright-dive performance (Wright, 1985). [Hb] and Hct Thermal sensitivity of fright-dive metabolism and metabolic levels rise in some vertebrates during hypoxia and/or diving (Thornton depression and Hochachka, 2004), probably stemming from recruitment of red Body temperature has long been identified as the most influential blood cells from the spleen (Hurford et al., 1985). This response was abiotic factor governing aerobic metabolism in ectotherms (Brett, not observed here and [Hb] and Hct remained unchanged following a ̇ 1971). VO2,pre-dive increased markedly between 28 and 34°C fright-dive, suggesting splenic contraction does not occur during (Q10=2.32), reflecting the typical metabolic response of predator avoidance dives in juvenile C. porosus. ectotherms to acute thermal increases (Schulte, 2015). Similar Q10 values have been reported for resting C. porosus over a comparable Error associated with estimating respiratory quotients temperature range (V̇ , Q =2.68, range=20–33°C; Grigg, Respiratory quotients (RQ) were estimated using an equation O2,standard 10 ̇ 1978), suggesting metabolic thermal sensitivity does not differ derived by Grigg (1978) for the majority of VO2 calculations and a between these contexts. In line with hypothesis 1, diving metabolic small margin of error may have been introduced into the absolute ̇ rate increased threefold between 28 and 34°C. This increase in values. The RQs were derived from VO2,standard measurements oxygen demand translates into a faster use of TBO stores during on the same species (i.e. C. porosus) with a similar body mass Journal of Experimental Biology

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(180–6200 g) and age ( juveniles) range, making estimates Competing interests comparable (Grigg, 1978). Using Grigg’s (1978) RQ assumes The authors declare no competing or financial interests. equal thermal sensitivity of V̇ , V̇ and V̇ . O2,standard O2,pre-dive O2,post-dive Author contributions This assumption may be valid as animals are stationary when resting Conceptualization: E.M.R., C.E.F.; Methodology: E.M.R., C.E.F.; Validation: E.M.R.; at the water surface, similar to resting conditions when measuring Formal analysis: E.M.R.; Investigation: E.M.R.; Resources: C.E.F.; Data curation: ̇ E.M.R.; Writing - original draft: E.M.R.; Writing - review & editing: E.M.R., C.E.F.; VO2,standard. Estimating RQs for conditions where anaerobic metabolism is partially fuelling activity, as may be the case here Visualization: E.M.R.; Supervision: C.E.F.; Project administration: C.E.F.; Funding with fright-dives, is often avoided as lactate accumulation can acquisition: C.E.F. shift the bicarbonate–CO equilibrium towards CO (Lighton and 2 2 Funding Halsey, 2011). However, post-dive recovery durations did not differ This work was supported in part by an Australian Research Council (ARC) Linkage between test temperatures, suggesting reliance on anaerobic Grant to C.E.F. and funding provided by The University of Queensland. metabolism (if any) was equal. Therefore, any error introduced from anaerobiosis would be the same and have no overall impact on Supplementary information the relative effect of test temperature on V̇ . Further reassuring Supplementary information available online at O2,dive http://jeb.biologists.org/lookup/doi/10.1242/jeb.155440.supplemental. is that the RQs calculated when the CO2 meter was functioning fall within the range measured by Grigg (1978; V̇ RQ=0.65± ̇ O2,pre-dive References 0.02, VO2,post-dive RQ=0.55±0.0, means±s.d.). Alboni, P., Alboni, M. and Gianfranchi, L. (2011). Diving Bradycardia: a mechanism of defence against hypoxic damage. J. Cardiovasc. Med. 12, 422-427. Ecological consequences of reduced fright-dive times Altimiras, J., Franklin, C. E. and Axelsson, M. (1998). Relationships between Fright-dive durations were reduced by ∼50% at a water temperature blood pressure and heart rate in the saltwater crocodile Crocodylus porosus. reflective of predicted climate change (i.e. 34°C), compared with J. Exp. Biol. 201, 2235-2242. present-day summer temperatures (28°C). This finding suggests Andersen, H. T. (1966). Physiological adaptations in diving vertebrates. Physiol. Rev. 46, 212-243. predator avoidance dives may be shortened if water temperatures Bellard, C., Bertelsmeier, C., Leadley, P., Thuiller, W. and Courchamp, F. continue to increase in marine and freshwater habitats. Additional (2012). Impacts of climate change on the future of biodiversity. Ecol. Lett. 15, concern stems from the finding that diving performance was equally 365-377. Bentivegna, F., Hochscheid, S. and Minucci, C. (2003). Seasonal variability in compromised at 34°C in crocodiles from both thermal acclimation voluntary dive duration of the Mediterranean loggerhead turtle, Caretta caretta. treatments (i.e. 28°C acclimated and 34°C acclimated). This finding Sci. Mar. 67, 371-375. demonstrates a lack of thermal phenotypic plasticity, which is thought Blix, A. S. and Folkow, B. (1983). Cardiovascular adjustment to diving in mammals to be a defining safeguard in long-lived species which may and birds. In Handbook of Physiology (ed. J. T. Shepard and F. M. Abboud), pp. 917-945. Bethesda: American Physiological Society. experience substantial thermal increases within a single lifetime. A Bradshaw, C. J. A., McMahon, C. R. and Hays, G. C. (2007). Behavioral inference non-existent thermal acclimation capacity (within 30 days) at of diving metabolic rate in free-ranging leatherback turtles. Physiol. Biochem. elevated temperatures has been demonstrated previously in Zool. 80, 209-219. Brett, J. R. (1971). Energetic responses of salmon to temperature. A study of some C. porosus (Rodgers et al., 2015). Similarly, many other diving thermal relation in the physiology and freshwater ecology of sockeye salmon ectotherms have limited thermal acclimation capacity (Graham et al., (Oncorhynchus mykiss). Am. Zool. 11, 99-113. 1971; Clark et al., 2008; Heatwole et al., 2012) and some species are Burggren, W. W. (1987). Form and function in reptilian circulations. Am. Zool. unable to compensate for seasonal thermal increases (Carr et al., 27, 5-19. Butler, P. J. (2006). Aerobic dive limit. What is it and is it always used appropriately? 1980; Bentivegna et al., 2003; Gordos et al., 2003; Hochscheid et al., Comp. Biochem. Physiol. A. 145, 1-6. 2005; Campbell et al., 2010a). Underlying this lack of acclimation Butler, P. J. and Jones, D. R. (1982). The comparative physiology of diving in capacity in the diving performance of C. porosus is probably the vertebrates. Adv. Comp. Physiol. Biochem. 8, 179-364. ̇ Butler, P. J. and Jones, D. R. (1997). Physiology of diving birds and mammals. inability to adjust rates of oxygen consumption (VO2,dive and Physiol. Rev. 3, 837-899. ̇ – VO2,pre-dive), fH, Hb and Hct to elevated temperatures as seen here. Campbell, H. A., Dwyer, R. G., Gordos, M. and Franklin, C. E. (2010a). Diving A lack of thermal acclimation capacity brings into question how through the thermal window: implications for a warming world. Proc. Roy. Soc. B. this species will fare as water temperatures in natural habitats 277, 3837-3844. Campbell, H. A., Sullivan, S., Read, M. A., Gordos, M. A. and Franklin, C. E. continually increase with climate change. Fright-dive durations will (2010b). Ecological and physiological determinants of dive duration in the probably decrease, with concomitant increases in surfacing freshwater crocodile. Funct. Ecol. 24, 103-111. frequency. Surfacing frequency has been shown to increase with Carr, A., Ogren, L. and McVea, C. J. (1980). Apparent hibernation by the Atlantic loggerhead turtle off Cape Canaveral, Florida. Biol. Conserv. 19, 7-14. rising water temperature in several diving ectotherms, including in Clark, N. J., Gordos, M. A. and Franklin, C. E. (2008). Thermal plasticity of diving sea snakes (Udyawer et al., 2016), marine and freshwater turtles behavior, aquatic respiration, and locomotor performance in the Mary River turtle (Southwood et al., 2003; Storey et al., 2008), newts (Šamajová and Elusor macrurus. Physiol. Biochem. Zool. 81, 301-309. ž Clark, T. D., Sandblom, E. and Jutfelt, F. (2013). Aerobic scope measurements of Gvo dík, 2009) and freshwater crocodiles (Campbell et al., 2010a). fishes in an era of climate change: respirometry, relevance and recommendations. Unless behavioural compensation is employed (e.g. diving in deep, J. Exp. Biol. 216, 2771-2782. cool water pockets or migrating polewards to cooler climates), Costa, D. P., Kuhn, C. E., Weise, M. J., Shaffer, S. A. and Arnold, J. P. Y. (2004). juvenile C. porosus may need to frequently replenish oxygen stores When does physiology limit the foraging behaviour of freely diving mammals? Int. Congress. Ser. 1275, 359-366. at the potential cost of becoming increasingly conspicuous to Davis, R. W., Polasek, L., Watson, R., Fuson, A., Williams, T. M. and Kanatous, S. B. predators. (2004). The diving paradox: new insights into the role of the dive response in air- breathing vertebrates. J. Comp. Biochem. Physiol. A. 138, 263-268. Douse, M. A. and Mitchell, G. S. (1988). Temperature effect on CO2-sensitive Acknowledgements intrapulmonary chemoreceptors in the lizard, Tupinambis nigropunctatus. Respir. We thank Dr Rebecca Cramp and Dr Craig White for advice and insightful Phyiol. 72, 327-341. discussions. Many thanks are extended to all volunteers who assisted with crocodile Drummond, P. C. and Jones, D. R. (1979). The initiation and maintenance of husbandry: D. Gomez Isaza, M. Elmer, C. Dudal, H. Goodrich, N. Wu, C. Ahlgren, bradycardia in a diving mammal, the muskrat, Ondatra zibethica. J. Physiol. 290, D. Stark and N. Clark. We are grateful to Jacqui Seal and the staff at David Fleay 253-271. Wildlife Park, and Matt Plummer and the staff at the Cairns Crocodile farm who made Eliason, E. J., Clark, R. D., Hague, M. J., Hanson, L. M., Gallagher, Z. S., this work possible by providing juvenile crocodiles and eggs. Jefferies, K. M., Gale, M. K., Patterson, D. A., Hinh, S. G. and Farrell, A. P. Journal of Experimental Biology

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